ON  THE  NECESSITY  OF  CULTI- 
VATING WATER  BACTERIA 
IN  AN  ATMOSPHERE  SAT- 
URATED WITH  MOISTURE 


By  GEORGE  C.  WHIPPLE,  S.B. 


Reprinted  from  Technology  Quarterly,  Volume  XII,  No.  4,  December,  1899 


MILLER’S 

White  Cloth  Flap  Binders, 

MANDFACT0RED  BT 


HAROLD  E.  MILLER 


SEND  FOR  PRICES. 


George  C.  Whipple . 


276 


ON  THE  NECESSITY  OF  CULTIVATING  WATER  BAC- 
TERIA IN  AN  ATMOSPHERE  SATURATED  WITH 

MOISTURE . 

By  GEORGE  C.  WHIPPLE,  S.  B. 

According  to  present  practice  the  number  of  bacteria  in  a sample 
of  water  is  found  by  mixing  a certain  quantity  of  the  water  with  a 
certain  quantity  of  sterilized  nutrient  gelatin  and  allowing  the  mixture 
to  solidify  in  a Petri  dish.  After  a longer  or  shorter  period  of  incuba- 
tion at  a temperature  at  or  about  20°  C.,  the  colonies  that  have  devel- 
oped upon  the  gelatin  are  counted,  and  from  this  count  the  number  of 
bacteria  present  in  the  water  is  determined.  Although  this  method 
is  almost  universally  used,  and  although  the  results  obtained  are  some- 
times of  the  greatest  moment,  there  is  such  a lack  of  uniformity  in 
the  details  of  the  process  as  ordinarily  conducted,  that  the  determina- 
tions of  different  observers  are  seldom  comparable  unless  the  methods 
of  procedure  are  fully  described.  A standard  method  of  procedure 
is  urgently  needed,  but  cannot  be  secured  until  the  various  factors 
that  influence  the  result  have  been  analyzed  and  their  magnitude 
determined.  In  the  course  of  a series  of  experiments  conducted  with 
this  object  in  view,  it  was  observed  that  the  amount  of  moisture  in 
the  atmosphere  of  the  incubator  exercised  an  important  influence  upon 
the  number  of  bacteria  that  developed.  The  results  of  these  obser- 
vations are  summarized  in  this  paper. 

Before  the  use  of  the  Petri  dish  it  was  customary  to  pour  the 
mixed  gelatin  and  water  upon  a cold  glass  plate,  where  it  was  allowed 
to  spread  out  and  harden.  The  plate  was  then  covered  with  a bell-jar 
and  put  in  the  incubator.  In  order  to  prevent  the  gelatin  from  drying, 
moist  filter  paper  was  put  at  the  bottom  of  the  bell-jar.  Thus  the  bac- 
teria* developed  in  a moist  atmosphere.  With  the  advent  of  the  Petri 
dish  the  matter  of  moisture  seems  to  have  been  lost  sight  of.  This 
dish  was  provided  with  a tight  fitting  cover,  and  this  was  supposed  to 
prevent  evaporation  from  the  gelatin.  It  is  a fact,  however,  that  the 
Petri  dishes  now  on  the  market  are  not  tight,  and  that  often  the  covers 


On  the  Cultivation  of  Water  Bacteria.  277 

fit  the  plates  very  badly.  Furthermore,  in  laboratories  where  many 
dishes  are  in  daily  use,  it  is  a common  thing  for  dishes  and  covers  to 
be  mismated,  and  often  there  is  no  attempt  to  mate  them.  The  result 
is  that  an  appreciable  evaporation  from  the  gelatin  does  take  place, 
and  that  the  amount  of  evaporation  varies  with  different  plates  and 
with  different  atmospheric  conditions. 

The  effect  which  this  uncontrolled  factor  exercises  upon  the  devel- 
opment of  bacteria  is  shown  by  the  following  experiments.  Several 
series  of  cultures  were  submitted  to  varying  conditions  of  moisture, 
with  other  conditions  remaining  constant.  The  plates  were  incubated 
in  a moist  chamber,  in  a closed  chamber  without  moisture,  in  the  incu- 
bator (or  the  ice  chest),  and  in  a desiccator.  For  the  moist  chamber  a 
desiccator-jar  was  used,  the  sulphuric  acid  being  replaced  by  water. 
The  closed  chamber  was  a desiccator-jar  without  sulphuric  acid  or 
water.  The  jars  were  large  enough  to  hold  five  plates,  and  in  all 
cases  the  figures  representing  the  numbers  of  bacteria  found  were 
obtained  from  the  averages  of  the  five  counts.  The  results  are  given 
in  the  following  tables.  Table  I shows  the  average  number  of  bac- 
teria that  developed  upon  each  set  of  plates.  In  Table  II  these  figures 
are  reduced  to  percentages  of  the  number  that  developed  in  the  moist 
chamber. 


TABLE  I. 


X — 

8 S2 

£ £ 
,H  4> 

■s.P 

Number  of  Bacteria  Per  Cubic  Centimeter. 

Series. 

Date. 

a a 
— 1 0 

O W 

T3  - 
O S 

•go 

Temperature  < 
cubation.  ( 
tigrade) 

In  moist 
chamber. 

In  ice  chest. 

In  incubator. 

In  closed 
chamber. 

In  desiccator. 

A. 

1899. 

July  7. 

96 

16° 

583 

527 





465 

B. 

July  7. 

96 

16° 

507 

430 

— 

— 

415 

C. 

July  7. 

96 

16° 

750 

683 

— 

— 

618 

D. 

Oct.  9. 

96 

15° 

450 

438 

— 

395 

342 

E. 

May  20. 

72 

18° 

154 

— 

115 

— 

111 

F. 

Oct.  14. 

72 

20° 

108 

— 

89 

90 

84 

G. 

Oct.  9. 

48 

20° 

423 

— 

413 

354 

324 

H. 

Oct.  17. 

72 

20° 

61 

— 

59 

— 

51 

2yS 


George  C.  Whipple. 


TABLE  II. 


ci  ^ 

■S? 
3 8 

S fl 

•r"  <D 

■sy. 

Per  Cent,  which  Each  Number  was  of  the  Number 
Found  in  the  Moist  Chamber. 

Series. 

Date. 

Period  of  i 
tion.  (H< 

Temperatur 

cubation. 

tigrade.) 

In  moist 
chamber. 

In  ice  chest. 

In  incubator. 

In  closed 
chamber. 

In  desiccator. 

A. 

1899. 

July  7. 

96 

16° 

100 

90 





79 

B. 

July  7. 

96 

1 6° 

100 

85 

— 

— 

82 

c. 

July  7. 

96 

16° 

100 

91 

— 

— 

82 

D. 

Oct.  9. 

96 

15° 

100 

98 

— 

88 

79 

E. 

May  20. 

72 

18° 

100 

— 

75 

— 

73 

F. 

Oct.  14. 

72 

20° 

100 

— 

82 

83 

77 

G. 

Oct.  9. 

48 

20° 

100 

— 

98 

83 

77 

H. 

Oct.  17. 

72 

20° 

100 

— 

97 

— 

83 

Average. 

100 

91 

88 

85 

78 

From  these  tables  it  will  be  seen  that  the  largest  numbers  of  bac- 
teria were  always  obtained  from  the  plates  that  developed  in  the  moist 
chamber,  and  that  -the  smallest  numbers  were  always  obtained  from 
the  plates  that  were  kept  in  the  desiccator.  If  the  number  that  devel- 
oped in  the  moist  atmosphere  be  assumed  to  represent  the  maximum 
number  obtainable  by  the  method,  it  follows  that  only  78  per  cent,  of 
the  bacteria  developed  in  the  desiccator,  that  85  per  cent,  developed 
in  the  closed  chamber,  and  that  the  numbers  that  developed  in  the 
ordinary  incubator  and  in  the  ice  chest  varied  from  75  per  cent,  to 
98  per  cent.  There  was  a striking  uniformity  in  the  percentages 
which  the  numbers  in  the  desiccator  were  of  the  numbers  in  the  moist 
chamber  and  this  was  due,  no  doubt,  to  the  constant  atmospheric  con- 
ditions in  the  two  jars.  In  the  closed  chamber  also  the  percentages 
were  quite  constant.  In  the  ice  chest  and  in  the  incubator  the  per- 
centages were  mqre  variable.  The  atmosphere  of  the  ice  chest  was 
ordinarily  more  moistM:han  that  of  the  incubator,  but  was  seldom  satu- 
rated on  account  of  ventilation.  The  amount  of  moisture  in  the  air 
of  the  incubator  was  generally  greater  than  that  of  the  atmosphere  of 
the  laboratory,  and  varied  more  or  less  with  the  ventilation,  the  number 
of  plates  kept  in  the  incubator,  etc.  In  Series  E the  relative  humidity 


On  the  Cultivation  of  Water  Bacteria.  279 

of  the  atmosphere  in  the  incubator  was  estimated  at  about  60  per  cent. 
In  Series  F the  humidity  was  determined  by  hourly  readings  of  a 
psychrometer  and  was  found  to  vary  between  65  per  cent,  and  80  per 
cent.,  the  average  being  75  per  cent.  In  Series  G the  atmosphere  of 
the  incubator  was  kept  moist  by  means  of  jars  of  water,  and  the 
average  humidity  was  95  per  cent.  In  Series  H the  humidity  was 
similarly  kept  at  98  per  cent,  of  saturation.  The  relation  between 
the  humidity  and  the  development  of  bacteria  is  shown  by  the  follow- 
ing comparison  : 


Series. 

Relative  Humidity  of  the  Atmosphere 
of  the  Incubator  in  per  cent,  of 
Saturation. 

Per  cent,  which  the  Number  of  Bacte- 
ria that  Developed  in  the  Incubator 
was  of  the  Number  that  Devel- 
oped in  the  Moist  Chamber. 

E. 

60  (estimated.) 

75 

F. 

75 

82 

G. 

95 

98 

H. 

98 

97 

The  amount  of  evaporation  from  gelatin  under  the  conditions 
described  above  was  determined  by  weighing  the  dishes  with  their 
contents  before  and  after  incubation.  It  was  found  that  in  a satu- 
rated atmosphere  the  evaporation  was  inappreciable ; in  the  closed 
chamber  it  was  from  1 to  2 per  cent,  of  the  weight  of  the  mixed 
water  and  gelatin  during  72  hours  ; in  the  ice  chest  it  was  about  3 
per  cent. ; in  the  incubator  it  was  from  3 per  cent,  to  5 per  cent. ; and 
in  the  desiccator  it  was  from  10  per  cent,  to  15  per  cent.  The 
amount  of  evaporation  varied  greatly  with  different  plates  submitted 
to  the  same  conditions. 

This  loss  of  water  by  evaporation  is  sufficient  to  cause  important 
changes  in  the  composition  of  the  culture  media  during  incubation, 
and  this  may  be  sufficient  to  affect  the  development  of  the  bacteria,, 
as  these  organisms  are  very  susceptible  to  slight  changes  in  environ- 
ment. But  the  chief  reason  why  increased  evaporation  is  accompa- 
nied by  a decrease  in  the  percentage  of  bacteria  that  develop  seems 
to  be  connected  with  the  supply  of  oxygen.  By  successive  weighing 
of  the  same  plates  it  was  found  that  the  rate  of  evaporation  was  not 
constant,  but  decreased  rapidly.  For  example,  during  the  first  hour 


28  o 


George  C.  Whipple. 


of  incubation  the  evaporation  from  a series  of  plates  proceeded  at  the 
rate  of  .068  gram  per  hour;  after  18  hours  the  rate  of  evaporation 
was  .031  gram  per  hour;  after  42  hours  it  was  .012  gram  per  hour, 
and  after  96  hours  it  was  .002  gram  per  hour.  This  retardation  of  the 
rate  of  evaporation  appears  to  be  due  to  a thickening  of  the  gelatin  at 
the  surface.  If  such  is  the  case,  the  thickened  gelatin  at  the  surface 
might  materially  reduce  the  supply  of  oxygen  of  the  submerged  bac- 
teria and  prevent  the  development  of  the  less  vigorous  aerobic  forms. 
Indeed,  it  was  observed  that  the  plates  in  the  desiccator  and  in  the 
moist  chamber  differed  more  in  the  number  of  submerged  colonies 
than  in  the  number  of  surface  colonies. 

These  facts  suggested  the  possibility  that  the  Petri  dish  itself 
might  serve  to  prevent  the  bacteria  from  getting  a sufficient  supply 
of  oxygen.  The  experiment  was  made,  therefore,  of  cultivating  the 
bacteria  in  a Petri  dish  with  the  ordinary  cover  replaced  by  a ground 
glass  top  that  made  an  absolutely  air  tight  joint.  A series  of  five 
cultivations  made  in  this  way  gave  the  number  of  bacteria  in  a certain 
sample  as  317  per  cc.,  while  a series  of  cultivations  of  the  same  water 
made  in  the  usual  manner  gave  413  per  cc.  In  the  hermetically 
sealed  dish,  therefore,  only  77  per  cent  of  the  bacteria  developed. 
The  air  of  these  dishes  was  then  collected  over  mercury,  and  the 
amount  of  oxygen  determined  by  absorption  with  pyrogallic  acid. 
It  was  found  that  the  air  of  the  ordinary  dishes  contained  approxi- 
mately 1 5 per  cent,  of  oxygen,  while  the  air  of  the  sealed  dishes  con- 
tained only  5 per  cent.  In  other  words,  three-quarters  of  the  original 
supply  of  oxygen  in  the  sealed  dishes  had  been  used  up  by  the  bac- 
teria during  72  hours,  while  one-quarter  of  the  original  supply  had 
been  used  up  in  the  ordinary  dishes.  It  was  found  also  that  the  air 
in  the  sealed  dishes  contained  5 per  cent,  of  C02,  while  the  air  in 
the  ordinary  dishes  contained  but  2 per  cent. 

As  a matter  of  interest  a series  of  cultures  was  made  in  a jar 
filled  with  oxygen,  and  compared  with  cultures  of  the  same  water 
made  in  the  incubator.  It  was  found  that  the  average  number  of 
colonies  that  developed  in  the  oxygen  was  1 54  per  cc.,  while  in  the 
incubator  it  was  137.  The  growth  in  oxygen  also  took  place  at  a 
much  more  rapid  rate. 

Finally,  one  important  fact  was  noted  in  connection  with  the  cul- 
tures made  in  the  jars  that  illustrates  the  important  effect  of  oxygen. 
In  all  the  jars  the  five  Petri  dishes  were  piled  one  on  top  of  the  other* 


On  the  Cultivation  of  Water  Bacteria. 


281 


and  it  was  found  that  almost  invariably  the  plate  highest  in  the  jar 
gave  the  largest  count,  and  that  the  lowest  plate  gave  the  smallest 
count.  This  was  particularly  striking  in  the  case  of  the  jar  filled  with 
oxygen,  where  the  upper  plate  contained  198  colonies  per  cc.,  while 
the  lower  plate  contained  138.  These  differences  between  the  upper 
and  lower  plates  of  the  jars  were  greater  than  the  differences  observed 
between  plates  kept  in  the  incubator.  Liquefaction  proceeded  much 
more  rapidly  in  the  upper  plates  of  the  jars.  These  phenomena 
seemed  to  be  due  to  an  accumulation  of  carbon  dioxide  at  the  bot- 
tom of  the  jars,  where  either  by  its  own  properties  or  by  exclusion 
of  oxygen,  it  retarded  the  growth  of  the  bacteria. 

These  facts  tended  to  show  that  the  supply  of  oxygen  has  a marked 
effect  upon  the  growth  of  water  bacteria  on  the  gelatin  plate,  and  that 
whatever  tends  to  reduce  the  supply  of  oxygen,  whether  it  be  evapo- 
ration or  an  air-tight  Petri  dish  or  lack  of  sufficient  ventilation,  will 
tend  also  to  reduce  the  number  of  bacteria.  The  practical  inference 
from  this  is  that  in  order  to  obtain  the  greatest  possible  develop- 
ment of  water  bacteria  on  the  gelatin  plate,  a ventilated  dish  should 
be  used,  and  the  cultures  should  be  incubated  in  an  atmosphere  satu- 
rated with  moisture. 

It  has  been  found  that  satisfactory  ventilation  of  the  Petri  dishes 
may  be  obtained  by  grinding  several  small  notches  in  the  edge  of  the 
lower  plate.  If  greater  ventilation  is  required,  it  may  be  obtained  by 
extending  the  sides  of  the  lower  plate  upwards  at  four  points  on  the 
circumference  so  as  to  form  pillars  for  supporting  the  cover,  thereby 
allowing  a complete  circulation  of  air.  It  has  been  found  that  the 
bacteria  counts  obtained  by  using  these  ventilated  dishes  in  an  atmos- 
phere saturated  with  moisture,  compare  closely  with  those  obtained  in 
open  dishes  protected  from  contamination  and  incubated  in  a saturated 
atmosphere,  the  results  of  one  series  of  comparisons  giving  97  and  95 
bacteria  per  cc,  respectively,  by  the  two  methods.  Experiments  have 
shown  that  there  is  no  danger  of  these  ventilated  plates  becoming 
contaminated  from  the  air. 

The  cultivation  of  water  bacteria  in  the  moist  atmosphere  has  the 
further  advantage  that  the  growths  come  to  maturity  in  a shorter  time. 
Liquefaction  takes  place  earlier,  but  the  growth  of  the  deep-seated 
colonies  seems  to  proceed  at  a still  more  rapid  rate.  A greater  pro- 
portion of  the  plates  are  liquefied  after  72  hours’  growth,  but  on  the 
other  hand,  the  percentage  increase  between  the  48-hour  count  and 
the  72-hour  count  is  much  reduced. 


282 


George  C.  Whipple. 


Incubators  used  for  the  cultivation  of  water  bacteria  should 
be  well  ventilated,  and  their  atmosphere  should  be  kept  at  or  near  the 
point  of  saturation.  They  should  be  provided  with  wet  and  dry  bulb 
thermometers,  and  the  relative  humidity  should  not  be  allowed  to  fall 
below  95  per  cent.  Experience  has  shown  that  an  atmosphere  prac- 
tically saturated  with  moisture  may  be  easily  maintained. 

Mt.  Prospect  Laboratory, 

Flatbush  Avenue  and  Eastern  Parkway, 

Brooklyn,  N.  Y. 


3 0112  072889071 


